Cytochrome P450 3A4 suppression by epimedium and active compound kaempferol leads to synergistic anti-inflammatory effect with corticosteroid.

Introduction: Cytochrome P450 (CYP) 3A4 is a major drug metabolizing enzyme for corticosteroids (CS). Epimedium has been used for asthma and variety of inflammatory conditions with or without CS. It is unknown whether epimedium has an effect on CYP 3A4 and how it interacts with CS. We sought to determine the effects of epimedium on CYP3A4 and whether it affects the anti-inflammatory function of CS and identify the active compound responsible for this effect. Methods: The effect of epimedium on CYP3A4 activity was evaluated using the Vivid CYP high-throughput screening kit. CYP3A4 mRNA expression was determined in human hepatocyte carcinoma (HepG2) cells with or without epimedium, dexamethasone, rifampin, and ketoconazole. TNF-α levels were determined following co-culture of epimedium with dexamethasone in a murine macrophage cell line (Raw 264.7). Active compound (s) derived from epimedium were tested on IL-8 and TNF-α production with or without corticosteroid, on CYP3A4 function and binding affinity. Results: Epimedium inhibited CYP3A4 activity in a dose-dependent manner. Dexamethasone enhanced the expression of CYP3A4 mRNA, while epimedium inhibited the expression of CYP3A4 mRNA and further suppressed dexamethasone enhancement of CYP3A4 mRNA expression in HepG2 cells (p < 0.05). Epimedium and dexamethasone synergistically suppressed TNF-α production by RAW cells (p < 0.001). Eleven epimedium compounds were screened by TCMSP. Among the compounds identified and tested only kaempferol significantly inhibited IL-8 production in a dose dependent manner without any cell cytotoxicity (p < 0.01). Kaempferol in combination with dexamethasone showed complete elimination of TNF-α production (p < 0.001). Furthermore, kaempferol showed a dose dependent inhibition of CYP3A4 activity. Computer docking analysis showed that kaempferol significantly inhibited the catalytic activity of CYP3A4 with a binding affinity of -44.73kJ/mol. Discussion: Inhibition of CYP3A4 function by epimedium and its active compound kaempferol leads to enhancement of CS anti-inflammatory effect.

Establishment of a predictive equation for pulmonary ventilation function in school-aged children in northeast China: a prospective study. / ä¸œåŒ—åœ°åŒºå­¦é¾„æœŸå„¿ç«¥è‚ºé€šæ°”åŠŸèƒ½é¢„è®¡æ–¹ç¨‹å¼å»ºç«‹çš„å‰çž»æ€§ç ”ç©¶.

OBJECTIVES: To establish a predictive equation for commonly used pulmonary ventilation function parameters in children aged 6-<16 years in northeast China. METHODS: A total of 504 healthy children from Liaoning, Jilin, and Heilongjiang provinces of China were selected for the prospective study, among whom there were 242 boys and 262 girls. The JAEGER MasterScreen Pneumo spirometer was used to measure pulmonary ventilation function. With the measured values of 10 parameters, including forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), FEV1/FVC ratio, and back-extrapolated volume (BEV), as dependent variables and age, body height, and body weight as independent variables, the stepwise multivariate regression method was used to establish the regression equation for children of different sexes. The mean of relative prediction error was used to evaluate the applicability of the predictive equation. RESULTS: The boys aged 9-<10 years and 15-<16 years had significantly higher body height, FVC, and FEV1 than the girls of the same age (P<0.05), and the boys aged 9-<10, 10-<11, 11-<12, and 13-<14 years had a significantly lower FEV1/FVC ratio than the girls of the same age (P<0.05). The correlation analysis showed that all parameters, except FEV1/FVC ratio and BEV/FVC ratio, were significantly positively correlated with age, body height, and body weight (P<0.001). Further regression analysis showed that age and body height were the influencing factors for most parameters, while body weight was less frequently included in the regression equation. Compared with the predictive equations from previous studies, the regression equation established in this study had relatively good applicability in the study population. CONCLUSIONS: A new predictive equation for the main pulmonary ventilation function parameters has been established in this study for children aged 6-<16 years in northeast China, which provides a basis for accurate judgment of pulmonary function abnormalities in clinical practice.

RET S409Y Germline Mutation and Associated Medullary Thyroid Carcinoma.

Background: Inherited medullary thyroid carcinoma (MTC) is primarily caused by RET mutations that are commonly localized in exons 5, 8, 10, 11, and 13-16. In this study, we report pedigrees for individuals with MTC that harbor a germline S409Y variant within exon 6 of the RET proto-oncogene. Methods: Targeted sequencing was used to diagnose four apparently sporadic MTC index cases carrying the germline RET S409Y (c.1226 C>A) variant. Subsequently, 27 relatives of these individuals underwent clinical and genetic assessments and/or thyroid surgery. Furthermore, in silico analyses and in vitro assays were performed to predict or verify the potential oncogenic activity of the S409Y variant. Results: Overall, 15 of 31 participants were found to carry the RET S409Y variant. Of these, 6 presented with isolated MTC (mean age 50.2 years; range 41-75 years), of which 3 presented with neck lymph node metastases and 2 presented with distant liver or lung metastases. Among the remaining 9 carriers, 3 (mean age 56 years; range 41-76 years) had elevated serum calcium-stimulated calcitonin (sCtn) or concurrent marginally elevated serum calcitonin (Ctn) levels, whereas the other 6 (mean age 37.5 years; range 14-52 years) exhibited typical Ctn/sCtn levels (p < 0.05). None of the 15 carriers in these 4 families presented clinical evidence of pheochromocytoma, hyperparathyroidism, or Hirschsprung's disease. In silico analyses revealed that S409Y was a "possibly damaging" mutation that could affect the RET protein inter-domain interface. An in vitro assay revealed that the phosphorylation level of RET tyrosine 905 was relatively higher in the RET S409Y mutant than in wild-type (WT) RET. Moreover, transfection of HEK 293 cells with S409Y enhanced the phosphorylation activity of AKT, ERK pathways, and it increased cell proliferation compared with WT RET, but to a lesser degree than that for the RET C618Y and C634Y mutations. Conclusions: This study demonstrates that the novel germline RET S409Y variant is likely pathogenic and is associated with lower penetrance of MTC than that for the C618Y and C634Y mutations. Individuals with S409Y should be managed using a personalized approach, and additionally, "at-risk" family members should be evaluated. Additional studies are needed to elucidate the correlation between the S409Y mutation and multiple endocrine neoplasia type 2-specific tumors.

The RET C611Y mutation causes MEN 2A and associated cutaneous

Background: Cutaneous lichen amyloidosis (CLA) has been reported in some multiple endocrine neoplasia type 2A (MEN 2A) families affected by specific germline RET mutations C634F/G/R/W/Y or V804M, as a characteristic of the clinical manifestation in 'MEN 2A with CLA', one of four variants of MEN 2A, which was strictly located in the scapular region of the upper back. Patient Findings: This study reports a large south-eastern Chinese pedigree with 17 individuals carrying the MEN 2A-harboring germline C611Y (c.1832G>A) RET mutation by Sanger sequencing. One individual presented MEN 2A-related clinical features, including typical CLA in the interscapular region; another individual exhibited neurological pruritus and scratching in the upper back but lacked CLA skin lesions. Both subjects presented with CLA or pruritic symptoms several years before the onset of medullary thyroid carcinoma (MTC) and/or pheochromocytoma. The remaining 15 RET mutation carriers did not exhibit CLA; of these, one presented with MTC and pheochromocytoma, nine with MTC only, two with elevated serum calcitonin and three younger subjects with normal serum calcitonin levels. This family's clinical data revealed a later diagnosis of MTC (mean age, 45.9 (range: 23­73) years), a lower penetrance of pheochromocytoma (2/17, 11.8%) and CLA (1/17, 5.9%). However, no hyperparathyroidism and Hirschsprung disease were reported in this family. Summary and Conclusions: This is the first description of a family with MEN 2A-related CLA due to a germline RET C611Y mutation, which might exhibit a novel and diversified genotype­phenotype spectrum in MEN 2A.

Immunotherapeutic effects of cytokine-induced killer cells combined with CCL21/IL15 armed oncolytic adenovirus in TERT-positive tumor cells.

The effective antitumor immune responses are dependent on coordinate interaction of various effector cells. Thus, the combination of adoptive immunotherapy and target gene therapy is capable of efficiently generating a productive antitumor immune response. We investigated whether combination of cytokine-induced killer (CIK) cells adoptive immunotherapy and CCL21/IL15 armed oncolytic adenovirus could induce the enhanced antitumor activity. The CCL21/IL15 co-expression oncolytic adenoviruses were constructed by using the AdEasy system, which uses homologous recombination with shuttle plasmids and full length Ad backbones. This conditionally replicating adenoviruses CRAd-CCL21-IL15 could induce apoptosis in TERTp-positive tumor cells for viral propagation, but do not replicate efficiently in normal cells, because the E1A promoter was replaced by telomerase reverse transcriptase promoter (TERTp). Our results showed that the combination of CIK cells and CRAd-CCL21-IL15 could induce higher antitumor activity than either CIK cells or CRAd-CCL21-IL15 alone. This combined treatment could induce the tumor specific cytotoxicity of CTLs (cytotoxic T lymphocytes) in vitro. Moreover, the treatment of established tumors with the combined therapy of CIK cells and CRAd-CCL21-IL15 resulted in tumor regression. This study suggests that the combined treatment by adoptive immunotherapy and gene therapy is a promising strategy for the therapy of tumor.

Tissue distribution and excretion study of neopanaxadiol in rats by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry.

Neopanaxadiol (NPD), a major ginsenoside in Panax ginseng C. A. Meyer (Araliaceae), was reported to have neuroprotective effect. In this study, a method of ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/QTOF-MS) was developed and validated for quantitative analysis of NPD in tissues, urine and feces, using liquid-liquid extraction (LLE) to isolate NPD from different biological samples, and chromatographic separation was performed on an Agilent Zorbax Stable Bond C18 (2.1 × 50 mm, 1.8 µm) column with 0.1% formic acid in water and acetonitrile. All standard calibration curves were linear (all r(2) > 0.995) within the test range. After oral administration, NPD was extensively distributed to most of the tissues without long-term accumulation. The higher levels were observed in stomach and intestine, followed by kidney and liver. Approximately 64.56 ± 20.32% of administered dose in feces and 0.0233 ± 0.0356% in urine were found within 96 h, which indicated that the major elimination route was fecal excretion. This analytical method was applied to the study of NPD distribution and excretion in rats after oral intake for the first time. The results we found here are helpful for us to understand the pharmacological effects of NPD, as well as its toxicity.

[Clinical diagnosis and treatment of familial medullary thyroid carcinoma caused by a p.C618Y RET proto-oncogene mutation in a Chinese pedigree].

OBJECTIVE: To explore the clinical characteristics, therapeutic and clinical significance for RET proto-oncogene screening in a pedigree with familial medullary thyroid carcinoma. METHODS: Comprehensive medical history was obtained from 19 members in a 4-generate southern Chinese family. Systemic clinical investigations including biochemical testing, imaging examinations and germline RET screening. RESULTS: RET screening showed heterozygous missense mutations of TGC to TAC at codon 618 on exon 10 in 8 cases (p.C618Y) completely consistent with the clinical manifestations. The clinical data of 7 patients with medullary thyroid carcinoma (MTC) and 2 carriers of asymptomatic RET mutation from were analyzed. Single/bilateral multi-centric MTC with lymph node metastases was confirmed in 6 cases by histopathology and 1 case by clinical examination. There were 1 male and 6 females with an initial mean diagnostic age was 49.6 years (range: 24 - 78). All had palpable neck masses. And the mean maximum diameter of MTC was 2.6 cm (range 1.4 - 4.4). Seven patients underwent thyroidectomy except a 78-year-old female patient: right total and left subtotal thyroidectomy (n = 1), right total thyroidectomy (previous left total thyroidectomy for benign mass) (n = 1) and total thyroidectomy (n = 4) were performed. All procedures were accompanied by at least bilateral level VI lymph node dissection and/or with modified single/bilateral neck dissection. After the first operation, 6 patients still presented a high value of calcitonin: 1 patient died of metastasis 64 months postoperatively; 3 patients underwent reoperation at 6 months after initial operation, the calcitonin levels dropped to normal in 2/3 cases and stayed higher in 1 case; another two cases presented bilateral thyroid gland residua, local lymph node enlargement on imaging examination and elevated levels of calcitonin at 214 and 60 months postoperation respectively. However, 1/2 asymptomatic with elevated pre-operative calcitonin subjects underwent total thyroidectomy and histopathological examination showed bilateral C cell hyperplasia. The other carriers, without surgery, with normal neck images, close monitoring and a 10-month follow-up, still presented undetectable calcitonin. CONCLUSIONS: Based on family survey, integrated RET screening and serum levels of calcitonin facilitate an early diagnosis and normalize surgery to improve the prognosis. For asymptomatic RET mutation carriers, their levels of calcitonin shall guide the individualized regimen of prophylactic thyroidectomy or strict monitoring and follow-ups.

Double contrast-enhanced two-dimensional and three-dimensional ultrasonography for evaluation of gastric lesions.

AIM: To investigate the value of two-dimensional (2D) and three-dimensional (3D) double contrast-enhanced ultrasonography (DCUS) imaging for evaluation of gastric lesions. METHODS: 2D and 3D DCUS imaging with both oral and intravenous administrations of contrast agents was used to assess gastroscopiclly-confirmed gastric lesions in 46 patients with benign and malignant diseases. Initially, liquid-based ultrasound contrast agent (Xinzhang®) was given orally at dose of 500-600 mL for conventional ultrasound examination of the gastric lesions, and then a microbubble-based contrast agent (SonoVue) was injected intravenously at dose of 1.2-2.4 mL in bolus fashion to assess the perfusion pattern of the lesions using contrast imaging modes. The parameters derived from time-intensity curves including the arrival time (AT), time to peak (TTP), peak intensity (PI) and enhanced intensity (EI) were measured on the 2D DCUS imaging. 3D DCUS of the lesions was acquired to demonstrate the value of this imaging mode. RESULTS: There were 22 cases with benign lesions including chronic gastritis (n = 5), gastric ulcer (n = 9), gastric polyps (n = 3), gastric stromal tumors (n = 5), and 24 cases with malignant lesions including gastric cancer (n = 20), gastric cardia carcinoma (n = 3) and post-operative recurrent gastric cancer (n = 1) in the study. The oral contrast-enhanced ultrasonography (CEUS) imaging of the stomach clearly demonstrated the anatomy of the stomach and morphologic features of gastric lesions. With optimal scanning window and imaging display under oral CEUS, intravenous CEUS clearly showed the perfusion of gastric lesions with various characteristic manifestations. Both 2D and 3D DCUS images clearly demonstrated normal gastric wall as a three-layer structure, from the inside out, hyperechoic mucosa, hypoechoic muscularis and hyperechoic serosa, respectively. There were statistical significant differences of AT (8.68 ± 2.06 vs 10.43 ± 2.75, P = 0.017), PI (34.64 ± 6.63 vs 29.58 ± 8.22, P = 0.023) and EI (29.72 ± 6.69 vs 22.66 ± 7.01, P = 0.001) between malignant lesions and normal gastric wall. However, no differences of AT, PI and EI between benign lesions and normal gastric wall tissue were found. 3D DCUS could intuitively display morphological features and vascularities of the lesions with multiplanar and volume views. 3D DCUS imaging provided comprehensive information complementary to 2D imaging. The crater or wellhead appearances and feeding vessels as well as distorted nourishing vasculature of gastric carcinoma were better seen with 3D imaging than 2D imaging. CONCLUSION: DCUS imaging can simultaneously display the anatomic and perfusion features of gastric lesions. 3D DCUS can provide additional information to 2D DCUS for evaluation of gastric lesions.

Repeated oral treatment with polysaccharide sulfate reduces insulin resistance and dyslipidemia in diabetic dyslipidemic rat model.

Polysaccharide sulfate (PSS) is a new type of antiatherosclerotic medicine for its effects of anticoagulation, anti-thrombosis and modulation of dyslipidemia. However, it is still uncertain whether PSS could modulate the diabetic dyslipidemia or not. Here, the rat model of diabetic dyslipidemia was developed and the effects of PSS on glucose and lipid levels were investigated in this animal model. Wistar rats were iv injected with streptozotocin 20 mg x kg(-1) after feeding with high fat diet for one and a half month. Then, rats received orally PSS (30, 90, and 180 mg x kg(-1)) for 1 month. After oral treatment with PSS (90 and 180 mg x kg(-1)) for 1 month, the levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) were significantly reduced and the level of high density lipoprotein-cholesterol (HDL-C) increased, compared with diabetic control rats. Moreover, PSS (30, 90, and 180 mg x kg(-1)) had a tendency to reduce glucose and insulin levels, and significantly increased insulin sensitivity index. Our results suggest that PSS could improve insulin sensitivity and relieve dyslipidemia in diabetic dyslipidemic rats.